Tuning the geometries of a de novo blue copper protein by axial interactions

• Published in: Journal of biological inorganic chemistry Vol. 17; no. 7; pp. 1025 - 1031
• Main Authors: Shiga, Daigo, Hamano, Yusuke, Kamei, Misato, Funahashi, Yasuhiro, Masuda, Hideki, Sakaguchi, Miyuki, Ogura, Takashi, Tanaka, Toshiki
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.10.2012
• Subjects: Amino Acid Sequence; Azides - chemistry; Binding Sites; Biochemistry; Biomedical and Life Sciences; Carrier Proteins - chemistry; Circular Dichroism; Color; Life Sciences; Ligands; Microbiology; Molecular Sequence Data; Original Paper; Protein engineering; 4-helix bundle protein; Metalloprotein; Blue copper protein
• ISSN: 0949-8257; 1432-1327
• DOI: 10.1007/s00775-012-0916-x

The axial interactions of Cu 2+ in type 1 copper proteins control the physical characteristics of the proteins. We tuned the geometries of a de novo designed blue copper protein with a four-helical bundle structure. The designed protein axially bound various ligands, such as chloride, phosphate, sulfate, acetate, azide, and imidazole, to Cu 2+ , exhibiting a blue or green color. The UV–vis spectral bands were observed at approximately 600 nm and approximately 450 nm, with the A ~450 / A ~600 ratios between 0.14 and 1.58. The stronger axial interaction shifted the geometry of the type 1 copper site from trigonal planar geometry (blue copper) toward a tetrahedral-like geometry (green copper). Resonance Raman spectral analyses showed that the phosphate-bound type had the highest-strength Cu–S bond, similar to that of plastocyanin. The chloride-bound type exhibited features similar to those of stellacyanin and nitrite reductase, and the imidazole-bound type exhibited features similar to those of azurin M121E mutant.